The long-term objective of this project is to elucidate the molecular basis for observed alterations in mitochondrial anion transporter function in type 1 (insulin-dependent) diabetes mellitus. Using the streptozotocin model of type 1 diabetes, we have recently discovered that the functional level of the liver mitochondrial citrate transporter is decreased and the levels of the pyruvate and dicarboxylate transporters are increased relative to control (i.e., nondiabetic) animals. Since these transporters are essential for the functioning of associated metabolic pathways (i.e., citrate transporter: fatty acid and sterol biosyntheses; pyruvate transporter: gluconeogenesis and fatty acid oxidation; dicarboxylate transporter: gluconeogenesis), and since the functioning of these metabolic pathways is altered in streptozotocin-- induced diabetes (i.e., hepatic fatty acid and possibly sterol biosyntheses are decreased, whereas gluconeogenesis and fatty acid oxidation are increased), we predicted that the level of mitochondrial anion transporter activity would be regulated in coordination with their associated metabolic pathways. Our initial studies support this hypothesis. We then showed that treatment of diabetic animals with insulin reversed the observed alterations in transporter function, which can thus be ascribed to the insulin deficiency that characterizes this disease. We now propose to elucidate the molecular basis for the observed altered function of the mitochondrial citrate and pyruvate transport proteins in type 1 diabetes. Specifically, utilizing liver mitochondria obtained from control, diabetic, and insulin-treated diabetic rats, experiments will be conducted to: 1) purify these transporters in reconstitutively active form and compare the intrinsic functional and molecular/chemical properties of the purified transport proteins; 2) quantify the amount of each transport protein present within the mitochondrial inner membrane; 3) determine the extent to which phosphorylation reactions regulate transporter function in vivo, in situ, and in vitro; and 4) determine the size, steady-state levels and rates of synthesis of transporter mRNAs. These studies will provide the first information concerning the molecular mechanisms by which mitochondrial transporter function is regulated in both the normal and the diabetic states. This information will then permit the future targeting of specific biochemical processes that regulate transporter function as possible sites for pharmacological intervention in diabetes.